Monomethyl Branched-Chain Fatty AcidsPlay an Essential Rolein Caenorhabditis elegans DevelopmentMarina Kniazeva1*, Quinn T. Crawford1, Matt Seiber1, Cun-Yu Wang2, Min Han1*1 Howard Hughes Medical Institute and Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado, United Statesof America, 2 Laboratory of Molecular Signaling and Apoptosis, Department of Biological and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor,Michigan, United States of AmericaMonomethyl branched-chain fatty acids (mmBCFAs) are commonly found in many organisms from bacteria tomammals. In humans, they have been detected in skin, brain, blood, and cancer cells. Despite a broad distribution,mmBCFAs remain exotic in eukaryotes, where their origin and physiological roles are not understood. Here we reportour study of the funct ion and regulation of mmBCFAs in Caenorhabditis elegans, combining genetics, gaschromatography, and DNA microarray analysis. We show that C. elegans synthesizes mmBCFAs de novo and utilizesthe long-chain fatty acid elongation enzymes ELO-5 and ELO-6 to produce two mmBCFAs, C15ISO and C17ISO. ThesemmBCFAs are essential for C. elegans growth and development, as suppression of their biosynthesis results in a growtharrest at the first larval stage. The arrest is reversible and can be overcome by feeding the arrested animals withmmBCFA supplements. We show not only that the levels of C15ISO and C17ISO affect the expression of several genes,but also that the activities of some of these genes affect biosynthesis of mmBCFAs, suggesting a potential feedbackregulation. One of the genes, lpd-1, encodes a homolog of a mammalian sterol regulatory element-binding protein(SREBP 1c). We present results suggesting that elo-5 and elo-6 may be transcriptional targets of LPD-1. This studyexposes unexpected and crucial physiological functions of C15ISO and C17ISO in C. elegans and suggests a potentiallyimportant role for mmBCFAs in other eukaryotes.Citation: Kniazeva M, Crawford QT, Seiber M, Wang CY, Han M (2004) Monomethyl branched-chain fatty acids play an essential role in Caenorhabditis elegans development.PLoS Biol 2(9): e257.IntroductionFatty acids (FAs) belong to a physiologically important classof molecules involved in energy storage, membrane structure,and various signaling pathways. Different FAs have differentphysical properties that determine their unique functions.Among the most abundant in animal cells as well as the moststudied are those of long-chain even-numbered saturated andunsaturated FAs.C15ISO and C17ISO are saturated tetradecano ic andhexadecanoic FAs with a single methyl group appended onthe carbon next to the terminal carbon (Figure 1). Mono-methyl branched-chain FAs (mmBCFAs) in ISO configurationas well as in anteISO configuration (methyl group appendedon the second to the terminal carbon) also seem to beubiquitous in nature. They are present in particularly largequantities in various bacterial genera, including cold-tolerat-ing and thermophilic species (Merkel and Perry 1977; Annouset al. 1997; Ferreira et al. 1997; Batrakov et al. 2000; Jahnke etal. 2001; Groth et al. 2002; Nichols et al. 2002). There,mmBCFAs contribute to the membrane function, regulatingfluidity (Rilfors et al. 1978; Suutari and Laakso 1994; Cropp etal. 2000; Jones et al. 2002) and proton permeability (van deVossenberg et al. 1999).Although comprehensive reports on mmBCFAs in eukar-yotes are lacking, sporadic data indicate that they are presentin the fungi, plant, and animal kingdoms (Garton 1985;Seyama et al. 1996; Martinez et al. 1997; Cropp et al. 2000;Wolff et al. 2001; Destaillats et al. 2002). In mammals,mmBCFAs have been detected in several tissues, includingskin (Aungst 1989), Vernix caseosa (Nicolaides and Apon 1976),harderian and sebaceous glands (Nordstrom et al. 1986), hair(Jones and Rivett 1997), brain (Ramsey et al. 1977), blood(Holman et al. 1995), and cancer cells (Hradec and Dufek1994). The fact that mmBCFAs are present in a wide variety oforganisms implies a conservation of the related metabolicenzymes and consequently important and perhaps uniquefunctions for these molecules (Jones and Rivett 1997).Nevertheless, their physiological roles and metabolic regu-lations have not been systematically studied and thus remainfragmentary. It was found that C21anteISO is the majorcovalently bound FA in mammalian hair fibers. A removal ofthis FA from its protein counterparts results in a loss ofhydrophobicity (J ones and Rivett 1997). Oth er stud iesindicated that C17anteISO esterified to cholesterol binds toReceived November 11, 2003; Accepted June 14, 2004; Published August 31,2004DOI: 10.1371/journal.pbio.0020257Copyright: Ó 2004 Kniazeva et al. This is an open-access article distributedunder the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.Abbreviations: ACC, acetyl-CoA carboxilase; BCAT, branched-chain aminotransfer-ase; BCKAD, branched-chain a-keto-acid dehydrogenase; CoA, coenzyme A; dsRNA,double-stranded RNA; FA, fatty acid; FAS, fatty acid synthetase; G3PA, glycerol 3-phosphate acyltransferase; GC, gas chromatography; GFP, green fluorescentprotein; mmBCFA, monomethyl branched-chain fatty acid; SI, saturation index;SREBP, sterol regulatory element binding proteinAcademic Editor: Paul W. Sternberg, California Institute of Technology*To whom correspondence should be addressed. E-mail: [email protected] (MK), [email protected] (MH)PLoS Biology | www.plosbiology.org September 2004 | Volume 2 | Issue 9 | e2571446Open access, freely available onlinePLoSBIOLOGYand activates enzymes of protein biosynthesis (Tuhackova andHradec 1985; Hradec and Dufek 1994). A potential signifi-cance of mmBCFAs for human health is associated with along-observed correlation between amounts of these FAs anddisease conditions such as brain deficiency (Ramsey et al.1977) and cancer (Hradec and Dufek 1994). More recentstudies have revealed a role of another mmBCFA, C15ISO, asa growth inhibitor of human cancer where it selectivelyinduces apoptosis (Yang et al. 2000). Given how importantthese FA molecules may be and how little is known abouttheir biosynthesis and functions in eukaryotes, it is anopportune problem to study.De novo synthesis of long-chain mmBCFAs described forbacteria is quite different from the